Fig. 2: ASS1 generates fumarate in the nucleus.
From: ASS1 metabolically contributes to the nuclear and cytosolic p53-mediated DNA damage response
a, Control and Dox-treated HCT116 WT cells were stained for ASS1 with Alexa Fluor 594 (red). n = 3 biologically independent experiments. b, Cytosolic and nuclear fractions of control and Dox-treated HCT116 WT cells were immunoblotted. Markers: lamin, nuclear; MEK, cytoplasmic Right panel: quantification of nuclear ASS1 normalized to H3 (Nuc), cytosolic ASS1 normalized to GAPDH (Cyto) and WCL ASS1 normalized to GAPDH (WCL). n = 4 biologically independent experiments. c, Livers from control (ASSFlox/Flox) and Alb-cre ASS1 mice (ASS1-D) were fractionated and immunoblotted for ASS1. Markers: H3, nuclear; GAPDH, cytoplasmic; OTC, mitochondrial. The faint band in cytosolic ASS1-D probably results from other liver cells expressing ASS1. n = 4 biologically independent samples. Right panel: quantification of nuclear ASS1 levels relative to H3 (Nuc) and cytosolic ASS1 levels normalized to GAPDH (Cyto). d, Control and Dox-treated HCT116 p53 WT and p53-KO cells were fractionated into cytosol (Cyto) and nuclear (Nuc) fractions and immunoblotted. Markers: H3, nuclear; tubulin, cytoplasmic; CPS1, mitochondrial. n = 5 biologically independent samples. Right panel: quantification of nuclear ASS1 levels relative to H3. e, ASS1–IPO7 interactions in control and Dox-treated HCT116 ASS1-EV and ASS1-KO cells. Right panel: quantification of total and nuclear puncta. n = 300 biologically independent cells across three biologically independent samples. f, Immunoprecipitation of IPO7 was performed on the nuclear fraction of control and Dox-treated HCT116 WT cells and immunoblotted (left). The nuclear lysate was immunoblotted for ASS1 and H3 (nuclear marker) (right). n = 3 independent experiments. g, Cytosolic and nuclear fractions of control and Dox-treated HCT116 WT cells transfected with scrambled or IPO7-targeted siRNA were immunoblotted for ASS1, p53 and IPO7. Markers: H3, nuclear; tubulin, cytoplasmic. Right panel: quantification of nuclear ASS1 protein expression levels, normalized to H3, and relative to ctrl. n = 5 independent experiments. Data are represented as the mean ± s.e.m. P values were determined by paired two-tailed Student’s t-test (in b and d), unpaired two-tailed Student’s t-test (in c and e) and two-tailed ratio paired t-test (in g). Boxplot in e represents min–max, with the line at the median and the shaded area representing the 50th percentile of each dataset.
